이 작품은 일본의 우에하라 기념 재단 (ES)을뿐만 아니라 화이트홀 재단, 신경 장애 및 뇌졸중과 스테인 - 오펜 하이머 엔다우먼트 수상 (BSK)을 국립 연구소에 의해 지원되었다.

실험 절차 실험 절차는 다음 단계 현명한 방식으로 설명하는 두 가지 핵심 부분으로 구성됩니다. 1 부 : 준비 HIPPOCAMPAL ASTROCYTE 문화 간단히 혼합 hippocampal astrocyte - 신경 세포의 문화가 잘 설립 프로토콜 1,2,3을 사용하여 준비되었습니다. 우리는 건강 교양 astrocytes를 얻을 수있는 절차를 최적화. 아래에 나열된 모든 절차는 이러한 층류 후드로 멸균 환경에서 수행되어야합니다. 준비 coverslips <ul><li> 22mm의 coverslips (VWR, 48380-068) </li><li> 폴리 - D - 라이신 (PDL, 시그마 P0899), aliquots (1 MG / ML) </li><li> Laminin aliquots가 (20 μg / ML) : 1 MG / ML laminin 솔루션 (시그마 L2020)에 멸균 물의 49 ML을 추가, -20 ° C에서 1.2 ML aliquots 및 저장을 </li></ul><ol><li> (50 μg / ML) 멸균 물의 PDL을 풀다 </li><li> coverslips을 압력솥, PDL의 살균 물과 장소 (100 coverslips 20 ML)로 헹굼. 하룻밤 상온에서 둡니다. </li><li> 멸균 물 (3 광범위한 세척)와 PDL을 바꿉니다. 그런 다음 4 coverslips하고 저장 ° C. 건조 </li><li> 6 잘 플레이트 (무균 Bioscience BD의 뚜껑과 Multiwell는 플랫 - 바텀 플레이트)에 대한 살균 잘에 astrocytes, 장소 개별 coverslips을 도금 전날. 각 coverslip에 laminin 400 μl를 넣고 증발을 방지하기 위해 인큐베이터에서 밤새 품어. </li></ol> 해부 해부 미디어 : <ul><li> 500 ML 얼의 밸런스드 소금 솔루션 (EBSS) (1X), 액체 (Invitrogen, 14155-063) </li><li> 5 ML HEPES 용액 (시그마, H0887) </li></ul> Hippocampal 미디어 : <ul><li> 412.5 ML 최소 필수 중간 (멤) (1X)은 액체가 얼의 염분을 포함하지만 L - 글루타민 또는 페놀 레드 없음 (Invitrogen, 51200-038) </li><li> 10 ML 포도당 (시그마, D - (+) - 포도당 무수 시그마 ULTRA G7528) 멤 1 M </li><li> 5 ML 페니실린 - 스트렙토 마이신 (Invitrogen, 15140-122) </li><li> 5 ML 나트륨 pyruvate 솔루션 (시그마, S8636) </li><li> 12.5 ML HEPES 용액 1 M (시그마, H0887) </li><li> 5 ML N - 2 보충 (100X), 액체 (Invitrogen, 17502-048) </li><li> 50 ML 호스 세럼, 열 Inactivated (Invitrogen, 26050-088) </li></ul> Papain 솔루션 : <ul><li> 워싱턴 PAPAIN - 022 유리병 (LK003178, 최종 농도가 20 U / ML)로 해부 미디어의 5 ML 추가 37 60 분 ° C 품어. </li></ul><ol><li> P1 (일반적으로이 새끼) 국가 규정 및 지역 기관 애니멀 케어 및 사용위원회 vetted 및 승인 지침에 따라 세 쥐 새끼 목을 벨. </li><li> 감기 해부 미디어 가득 배양 접시에있는 피부와 두개골과 장소의 두뇌를 제거합니다. </li><li> meninges을 제거, 두 반구를 해부하다하고 hippocampi을 해부하다. </li><li> 37 papain 솔루션 ~ 1X1 mm 조각 소화 ° C 11-13 분에 hippocampi 잘라. </li><li> 조직의 조각이 정착되면, 제거 신중하게 papain과 효소의 모든 흔적을 제거하는 hippocampal 매체의 5 ML를 추가합니다. hippocampal 매체 (2 ML)에서이 단계와 resuspend를 반복합니다. </li><li> 점차적으로 더 작은 한거죠 (1000 μm의 ~ 500 μm의와 ~ 300 μm의)의 세 불꽃 - 광택 pipettes로 남아 대단히 짧은 시간은 없습니다 때까지 세포 ~ 5 번 씹다. </li><li> 일단 triturated, 셀 스트레이너 (기공 크기, 70 μm의, BD Bioscience)를 통해 세포를 전달합니다. 정지 10 μl의 세포를 계산합니다. 400000 세포 / ML을 위해 hippocampal 매체의 볼륨을 조정합니다. </li><li> coverslips에서 laminin을 기음과 coverslip 당 세포 현탁액의 표지가 건조하기 전에 판 200 μl. </li><li> 그들이 인큐베이터에서 60 분 부착 후 잘 당 hippocampal 중간 2 ML을 추가할 수 둡니다. </li><li> 다음 날, 죽은 세포와 찌꺼기를 제거하고 미리 예열 신선한 hippocampal 중간 2 ML을 추가하려면 이전 hippocampal 매체를 대기음. </li></ol> 유지 Neurobasal 미디어 : <ul><li> 500 ML neurobasal (Invitrogen, 12348-017) </li><li> 10 ML B27 혈청 무료 보충 (Invitrogen, 17504-044) </li><li> 5 ML 페니실린 - 스트렙토 마이신 (Invitrogen, 15140-122) </li><li> 1.25 ML L - 글루타민 (Invitrogen, 25030-149) </li></ul> 도금 후 사일 시작 neurobasal 매체와 astrocyte - 뉴런 문화 일주일에 두 번 먹이. 온도와 CO 2를 평형에 통풍이 플라스크에 인큐베이터에서 30 분 대한 미디어 Preincubate. 2 부 : 칼슘 이미징 Hippocampal 레코딩 버퍼 : 110 MM NaCl, 5.4 MMKCl, 1.8 MM CaCl 2, MgCl 2 0.8 MM 10 MM D - 글루코오스, 10 MM HEPES (시그마의 모든 화학 물질) 산도 7.4 (NaOH로 조정). astrocytes에 칼슘 지표 염료를 로딩 <ol><li> (Invitrogen, DMSO 2.5 μm의 Fluo - 4 AM (Invitrogen, F - 14217)와 0.05 %의 Pluronic ® F - 127 20 % 용액을 포함하는 두 ML hippocampal 기록 버퍼로 가득한 6 잘 접시에 잘있는 문화 플레이스 P - 3000MP)와 10-30 분 실온에서 알을 품다. </li><li> Fluo - 4 솔루션을 제거하고 30 분 실온에서 hippocampal 기록 버퍼와 부화와 coverslips에게 3 회 씻는다. </li><li> 챔버에 coverslip를 삽입하고, 렌즈 클리닝 액체로 coverslip의 다른 측면을 청소. </li><li> 목적에 침지 기름의 작은 방울 (Cargille에서 집중 오일 종류 DF)를 넣고 현미경의 무대 (올림푸스 IX71)에 실 (워너 인스 트루먼 트에서 25mm의 원형 coverslip에 대한 열기 챔버)을 넣어. </li><li> 세포가 보이는지, 그리고 초점에 그들을 위해 전송 라이트를 사용하여 세포보세요. 다음 (비전까지에서 폴리 크롬 V) 단색에서 488 nm의와 세포를 조명하고 Fluo - 4의 형광 신호가 균일하고 astrocytes에 감지할 수 있는지 확인합니다. </li><li> astrocytes에 칼슘을 측정하는 에피네프린과 TIRF 조명을 사용합니다. </li></ol> TIRF 현미경 간단히, 우리는 Andor IXON DV887DCS EMCCD 카메라가 장착된 올림푸스 현미경 IX71을 사용합니다. 여기 및 이미지 수집의 제어 TILLVision 소프트웨어를 사용하여 이루어진다. 454/488/515 nm의 아르곤 (100 MW)와 442 nm의 고체 (45 MW) 레이저의 광선이 결합과 함께 제어까지 폴리 라인 레이저 결합기, TIRF 듀얼 포트 콘덴서와 acoustoptical tuneable 필터와 컨트롤러 (AOTF, 모든의 TIRF의 콘덴서에 입국 Kineflex 넓은 밴드 섬유로 Photonics) 및 공급 때까지. 우리는 TIRF를 달성하는 올림푸스 60X 1.45 NA 렌즈를 사용합니다. 카메라 이득은 노이즈 이미지를 최상의 신호를 제공하는 각 astrocyte에 대해 조정됩니다. 배경 및 TIRF 현미경의 원리는 최근 5 4을 검토하고 있습니다. 우리가 사용하는 광학 부품의 대부분은 이제 애질런트 테크놀 로지스 (http://www.till-photonics.com/)의 일부 Photonics,까지에서 구입한했다. 티르의 침투 깊이는 아래의 방정식으로부터 계산하실 수 있습니다. <img style="float:left;" src="/files/ftp_upload/1142/equation.png" alt="방정식" /> D = 침투 깊이 유리 N1 =의 굴절률 세포의 N2 = 굴절률 발병률의 A = 각도 발병률의 나이 = 숫자 조리개 레이저가 TIRF을위한 최적의 정렬되도록하기 위해 우리는 100 nm의 형광 구슬 (Invitrogen, F8803)를 준수하는 것이 유용할. 우리는 여전히 프레임과 에피와 TIRF 현미경과 구슬의 동영상을 제시한다. TIRF 경우에, 하나는 신호 대 잡음의 극적인 증가를 관찰하고 구슬이 Brownian 확산을 표시합니다. 중요한 각도가 동일하지 않다면 우리는 최적의 TIRF 오히려 발생할 위험 경사 조명보다 발생 있는지 확인하기 위해 정기적으로 (~ 한 주당)에 TIRF 현미경 100 nm의 비즈의 행동을 관찰하는 것이 유용 α (그림 1 참조). G - 단백질 결합 수용체 agonists의 응용 Astrocytes는 metabotropic 글루 탐 산염 수용체와 P2Y 수용체 (작용제, ATP, ADP)를 포함 GQ - 결합 수용체 6, 7의 다양한 표현한다. 이들 수용체의 활성화는 astrocytes 이내 세포 내 칼슘 수준에서 크게 증가 발생합니다. 예를 들어, 하나는 쉽게 astrocytes의 80-10에 ATP (30 μm의)의 응용 프로그램을하는 동안 세포 내 칼슘의 고도를 볼 수 있습니다. 우리는 워너 인스 트루먼 트에서 스위처는 VC - 77SP 빠른 단계 재관류 시스템 (http://www.warneronline.com/index.cfm를)라는 빠른 솔루션을 사용합니다. 이 방법 솔루션은 ~ 10 MS 이하에 적용할 수로. <img src="/files/ftp_upload/1142/Fig 1a.jpg" alt="그림 1" /> <img src="/files/ftp_upload/1142/fig1c.jpg" alt="" /> 그림 1. 만화와 영상의 사진가 설정할 수 있습니다. (B) 레이저 어셈블리, 컨트롤러 및 빔 상자의 사진을 보여주는 반면 A.는 airtable에 장착된 현미경의 사진을 보여줍니다. C.이 영상에 대한 마운트 챔버와 함께 현미경 단계의 사진을 보여줍니다. 에 빠른 재관류 장치 (스테퍼 모터와 세타 튜브와 함께)을 볼 수 있습니다 떠났다. 오른쪽 Axopatch 200A 증폭기의 headstage 볼 수 있습니다. 만화는 세트에서 빛을 경로를 schematizes 어떻게 TIRF가 이루어집니다. 레이저는 그것이 중요한 각도 α에 침지 기름에 나온다 있도록 중심을 조정하는 60X 1.45 NA 목표 렌즈와 위치의 뒤쪽 초점 비행기에 초점을두고 있습니다. 이 시점에서 빔 낮​​은 굴절률의 중간에 거리 λ (주 텍스트로 방정식을 참조) 총 내부 반사와 자연 붕괴로 인해 고통받는. 이 경우에 이것은 astrocytes 및 astrocytes 자신을 둘러싼 녹음 버퍼입니다. 결과는 플라즈마 막의 ~ 100 nm의 시간 분자의 광학 여기 (때문에 영상)입니다. 세포 내에서 또는 세포의 상단 표면에 사람들이 흥분되지 않은 반면, 세포의 만화는 이것이 녹색 &quot;흥분&quot;막 수용체로 표시됩니다. TIRF 현미경의 전체 계정은 스테 이어와 Almers 사에 의해 제공되었습니다. <img src="/files/ftp_upload/1142/fig2.png" alt="그림 2" /> 그림 2. 에피와 TIRF 현미경과 함께 인수 100 nm의 형광 구슬의 이미지. A. 여러 개 100 nm의 형광 구슬로보기의 필드의 에피 이미지를 표시합니다. 파란 화살촉이 물에 diffusing 아르 구슬을 가리킨 반면, 유리 coverslip에 정착 구슬에 빨간색 화살표 지점. B.는 빨간색 화살표로 표시된 자기편 비즈가 표시되는이보기에서는 A. 같이보기 같은 분야의 TIRF 이미지를 표시합니다. 이들은 유리 coverlsip에 정착하고 ~ 100 nm의 지극히 미미한 분야 내에서 따라서 이었어요 때문입니다. 파란 화살표로 표시 구슬이 지역 내에서되지 않으며 TIRF 이미지에 따라서 보이지 않습니다. 낮은 줄거리는 이미지의 3D 렌더링을 보여줍니다. 그것은 TIRF 현미경으로 관찰하면 신호 대 잡음의 큰 증가가 지극히 미미한 분야 내에서 구슬에 대한 발생 분명하다. TIRF 위해 20되었다 반면 ± 0.7 사실 이러한 이미지에 대한 에피네프린을위한 신호 대 잡음은 7.1 ± 0.6되었다. <img src="/files/ftp_upload/1142/fig3.png" alt="그림 3" /> 그림 3. 에피와 TIRF 현미경과 함께 인수 Fluo - 4 칼슘 표시기 색소와 함께 로드된 이미지 astrocytes. 오 astrocytes 전망이 분야의 A. 에피 이미지. 의 이미지가 A와 B가 크게 다르다는 것을 B. 참고보기에 표시된 것과 같은 분야의 B. TIRF 이미지. TIRF 조명과 유리 coverslip에 가까운 동격에서만 플라즈마 막 지역이 몇 군데 있기 때문입니다. 하단 패널은 표시 ATP - evoked 에피와 TIRF 현미경과 몇 군데 세포내 칼슘 과도합니다.

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N., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+forms+of+astrocyte+single+vesicle+exocytosis+imaged+with+total+internal+reflection+fluorescence+microscopy.">Two forms of astrocyte single vesicle exocytosis imaged with total internal reflection fluorescence microscopy.</a> Proc Natl Acad Sci U S A. 104, 4212-4217 (2007).</li><li>Bowser, D. N., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+ATP+is+the+predominant+cause+of+intercellular+calcium+waves+in+astrocytes.">Vesicular ATP is the predominant cause of intercellular calcium waves in astrocytes.</a> J Gen Physiol. 129, 485-491 (2007).</li><li>Agulhon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+role+of+astrocyte+calcium+in+neurophysiology.">What is the role of astrocyte calcium in neurophysiology.</a> Neuron. 59, 932-946 (2008).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60, 430-440 (2008).</li><li>Lee, S. Y., Haydon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytic+glutamate+targets+NMDA+receptors.">Astrocytic glutamate targets NMDA receptors.</a> J Physiol. 581, 887-888 (2007).</li><li>Shigetomi, E., Bowser, D. N., Sofroniew, M. V., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+forms+of+astrocyte+calcium+excitability+have+distinct+effects+on+NMDA+receptor-mediated+slow+inward+currents+in+pyramidal+neurons.">Two forms of astrocyte calcium excitability have distinct effects on NMDA receptor-mediated slow inward currents in pyramidal neurons.</a> J Neurosci. 28, 6659-6663 (2008).</li><li>Cahoy, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptome+database+for+astrocytes,+neurons,+and+oligodendrocytes:+a+new+resource+for+understanding+brain+development+and+function.">A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function.</a> J Neurosci. 28, 264-278 (2008).</li></ol>

그것은 astrocytes은 세포 내 칼슘의 고도를 표시 잘 설정됩니다. 이들은 자발적으로 발생하는가의 연결 활동이나 astrocyte 표면 11 수용체를 활성화하기 위해 agonists의 응용 프로그램에 의해 실행하실 수 있습니다. 하나의 중요한 논쟁 문제는 astrocyte 세포 내 칼슘의 고도는 뉴런 11, 12 수용체를 활성화 신호 분자의 릴리스를 실행할 수 있는지 여부입니다. 로 헤이든 7, 13, 11 맥카시 실험실?...

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<td>VWR&#174; Micro Cover Slips, Round, No. 1</td>
<td>Tool</td>
<td>VWR</td>
<td>48380-068</td>
<td>&nbsp;</td>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P0899</td>
<td>&nbsp;</td>
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<td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L2020</td>
<td>&nbsp;</td>
<tr>
<td>Earle&#x2019;s Balanced Salt Solution (EBSS) (1X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14155-063</td>
<td>&nbsp;</td>
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<td>Minimum Essential Medium (MEM) (1X), liquid Contains Earle's salts, but no L-glutamine or phenol red</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>51200-038</td>
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<td>Penicillin-Streptomycin liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15140-122</td>
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<td>Sodium pyruvate solution</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8636</td>
<td>&nbsp;</td>
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<td>HEPES solution 1 M</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H0887</td>
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<td>N-2 Supplement (100X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17502-048</td>
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<td>Horse Serum, Heat-Inactivated</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>26050-088</td>
<td>&nbsp;</td>
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<td>PAPAIN-022</td>
<td>Reagent</td>
<td>Worthington</td>
<td>LK003178</td>
<td>&nbsp;</td>
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<td>Neurobasal&#x2122; Medium (1X) Liquid without Phenol Red</td>
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<td>Invitrogen</td>
<td>12348-017</td>
<td>&nbsp;</td>
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<td>B-27 Serum-Free Supplement (50X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17504-044</td>
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<td>L-Glutamine-200 mM (100X), liquid</td>
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<td>Invitrogen</td>
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<td>Cell Strainers</td>
<td>Tool</td>
<td>BD Biosciences</td>
<td>352350</td>
<td>&nbsp;</td>
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<td>BD Falcon Multiwell Flat-Bottom Plates with Lids, Sterile</td>
<td>Tool</td>
<td>BD Biosciences</td>
<td>353046</td>
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<td>NaCl</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
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<td>Sigma</td>
<td>P3911</td>
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<td>Reagent</td>
<td>Sigma</td>
<td>21108</td>
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<td>Reagent</td>
<td>Sigma</td>
<td>M2670</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
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<td>D-(+)-glucose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G7528</td>
<td>&nbsp;</td>
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<td>Fluo-4, AM 1 mM solution in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F-14217</td>
<td>&nbsp;</td>
<tr>
<td>Pluronic&#174; F-127 20% solution in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>P-3000MP</td>
<td>&nbsp;</td>
<tr>
<td>Immersion Oil TYPE DF</td>
<td>Microscope</td>
<td>Cargille</td>
<td>16242</td>
<td>&nbsp;</td>
<tr>
<td>Open chamber for 25 mm round coverslips, 100 &mu;l volume</td>
<td>Tool</td>
<td>WARNER Instruments</td>
<td>64-0362 (RC-21BDW)</td>
<td>&nbsp;</td>
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<td>P-2 platform for Series 20 chambers, non-heater</td>
<td>Tool</td>
<td>WARNER Instruments</td>
<td>64-0278 (P-2)</td>
<td>&nbsp;</td>
<tr>
<td>FluoSpheres carboxylate-modified microspheres, 0.1 &mu;m, yellow-green fluorescent (505/515) 2% solids</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F8803</td>
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measuring near plasma membrane and global intracellular calcium dynamics in astrocytes

두뇌는 glial 세포가 포함되어 있습니다. Astrocytes, glial 세포의 종류는 긴 뉴런에 수동 지원 역할을 제공하는 것으로 알려져 있습니다. 그러나, 증가 증거 astrocytes도 적극적으로 뉴런과 기능적 상호 작용을 통해 뇌의 기능에 참여할 수있다하는 것이 좋습니다. 그러나, astrocyte 생물학의 많은 근본적인 측면 논쟁, 불분명 및 / 또는 실험적으로 미개척 남아있다. 중요한 문제는 astrocytes의 세포내 칼슘 과도의 역학입니다. 칼슘이 잘 중요한 두번째 메신저로 설립되고 있기 때문에 그것이 astrocyte 칼슘 고도가 astrocytes에서 송신기의 릴리스를 실행할 수있는 제안 되었기 때문에이 적합합니다. 그러나, astrocytes의 신호 근처 플라즈마 막 칼슘의 자세한 설명을하거나 만족이되지 않았습니다. 총 내부 반사 형광 (TIRF) 현미경 살 세포의 플라즈마 막의 약 100 nm의 시간 생리학 관련 신호 이벤트를 분석하는 강력한 도구입니다. 여기, 우리는 TIRF 현미경을 사용하여 거의 동시에 플라즈마 막과 글로벌 세포내 칼슘 역학 근처 모니터링하는 방법에 대해 설명합니다. 이 방법의 자세한 상세하고 체계적인 응용 프로그램은 astrocyte 칼슘 신호의 정확한 세부 사항에 대해 알리기 위해 가능성이 있습니다. astrocyte 칼슘 역학에 대한 상세한 이해, 어떻게 언제 왜 astrocytes와 뉴런은 칼슘 의존 기능 상호 작용을 거쳐, 이해하는 기초를 제공할 수 있습니다.

Watch this Scientific Journal Video about Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes at JoVE.com

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

우리는 총 내부 반사와 epifluorescence 현미경을 사용하여 교양 astrocytes에 막 및 글로벌 세포내 칼슘 역학 근처 측정하는 방법에 대해 설명합니다.

Astrocytes의 플라즈마 막 글로벌 세포내 칼슘 역학 니어 측정

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

measuring-near-plasma-membrane-global-intracellular-calcium-dynamics

Research

JoVE Journal

Biology

13.0K 조회수.  David Geffen School of Medicine, University of California, Los Angeles. 우리는 총 내부 반사와 epifluorescence 현미경을 사용하여 교양 astrocytes에 막 및 글로벌 세포내 칼슘 역학 근처 측정하는 방법에 대해 설명합니다.

비디오: Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

가역 Biotinylation에 의해 플라즈마 막 단백질 Endocytic 요금 측정

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

연구

생물학

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes의 빠른 칼슘 Fluxes 측정

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

다중 타겟 추적 (MTT)에 의해 플라즈마 막의 분자 확산을 매핑

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

형광 단백질을 표현 라이브 마우스의 이미징 세포 이하의 구조물의 intravital 현미경

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

pTIRFM와 이미징 플라즈마 막 변형

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

나의 기능적 특성<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt; 선택을 양자가 살해 사용하여 세포 내 구획의 교환기는 플라즈마 막에서 그들을 표현하는

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

원형질 막 주변 관련 된 단백질에 대 한 분할 결정

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.

Necroptosis에서 MLKL-중재 원형질 막 파열의 특성

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

세포내 칼슘과 갓 고립과 그대로 마우스 대뇌 피에 막 잠재력의 동시 측정

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

마우스 췌도 세포의 하위 집단에 있는 화상 진찰 칼슘 역학

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...